The present invention relates to methods for identifying agents capable of inhibiting the expression or activity of proteins involved in the processes modulating osteoclastogenesis, which inhibition is useful in the prevention and/or treatment of bone and joint degenerative diseases and diseases involving aberrant activity of osteoclasts. In particular, the present invention provides methods for identifying agents for use in the prevention and/or treatment of rheumatoid arthritis.
Rheumatoid arthritis (RA) is a chronic joint degenerative disease, characterized by inflammation and destruction of the joint structures. When the disease is unchecked, it leads to substantial disability and pain due to the loss of joint functionality and even premature death. The aim of an RA therapy, therefore, is not to slow down the disease but to attain remission in order to stop the joint destruction. Besides the severity of the disease outcome, the high prevalence of RA (˜0.8% of adults are affected worldwide) means a high socioeconomic impact (For reviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt (2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).
Histological analysis of the joints of RA patients clearly evidences the mechanisms involved in the RA-associated degradative processes. The synovium is a cell layer, composed of a sublining and a lining region that separates the joint capsule from the synovial cavity. The inflamed synovium is central to the pathophysiology of RA. The synovial joint is shown as composed of two adjacent bony ends each covered with a layer of cartilage, separated by a joint space and surrounded by the synovial membrane and joint capsule. The synovial membrane is composed of the synovial lining (facing the cartilage and bone), which consists of a thin (1-3 cells) layer of synoviocytes and the sublining connective tissue layer that is highly vascularised. Histological differences in the synovium between normal and RA patients are indicated in FIG. 1.
Like many other forms of arthritis, rheumatoid arthritis (RA) is initially characterized by an inflammatory response of the synovial membrane (‘synovitis’) that is characterized by an important influx of various types of mononuclear cells as well as by the activation of the local or infiltrated mononuclear cells. The lining layer becomes hyperplastic (it can have a thickness of >20 cells) and the synovial membrane expands. However, in addition, the hallmark of RA is joint destruction: the joint spaces narrow or disappear as a sign of cartilage degradation and destructions of the adjacent bone, also termed ‘erosions’, have occurred. The destructive portion of the synovial membrane is termed ‘pannus’. Various forms of bone degradation are apparent in RA. Besides a generalized osteoporosis, RA is also characterized by the erosion of the bone under and adjacent to the cartilage. These focal erosions result principally from the presence of an increased population of osteoclasts at the interface of bone and pannus (for a review on bone degradation in RA, we refer to Gravallese, 2002). Osteoclasts are multinucleated cells that attach to bone and secrete bone matrix degrading enzymes (e.g. Cathepsin K, MMP9) in an acidified space between the cell and the bone tissue (the resorption lacuna). In healthy individuals, the remodeling of bone is controlled by the activity of these osteoclasts, which resorb bone, and the activity of osteoblasts, which are involved in the production of the calcified bone matrix. Osteoblasts differentiate from mesenchymal stem cells, while osteoclasts differentiate from hematopoietic monocyte/macrophage precursors.
In RA, the concentration of the factors inducing osteoclast differentiation is increased at the interface between bone and the pannus (Pettit et al., 2006), leading to the dysregulation of the balance between bone formation and bone degradation. Key players in osteoclast differentiation are the receptor activator of NF-κB (RANK) and its ligand (RANKL) and osteoprotegerin (OPG).
RANKL is a membrane-anchored ligand of the TNF superfamily. In normal bone tissue, RANKL is expressed by osteoblasts, but in RA, synovial fibroblasts as well as activated T lymphocytes are important sources of RANKL. RANKL exerts its effect on osteoclasts or osteoclast precursor cells through RANK, a member of the TNF receptor superfamily. Another key player in osteoclast biology is OPG, a RANKL decoy receptor, which belongs to the TNF receptor superfamily and competes with RANK for the binding of RANKL. OPG, therefore, effectively inhibits osteoclast maturation and osteoclast activation. OPG-transgenic mice have a high bone mass (osteopetrosic phenotype), whereas the absence of OPG results in severe osteoporosis, as shown in OPG-knockout mice (Bucay et al., 1998). In summary, the balance between RANK/RANKL signaling and levels of OPG, the soluble decoy receptor for RANKL, regulates the development and activation of osteoclasts and therefore is strongly involved in bone metabolism. Thus, inhibition of RANKL function via OPG might prevent bone destruction in several diseases, e.g., RA. Of significance in this respect is the observation that RANKL knock-out mice are less prone to bone erosion when subjected to CIA (Pettit et al., 2001) and that recombinant OPG, alone or in combination with an anti-TNFα, prevents bone erosions in animal models for RA (Redlich et al., 2004). In addition, the capacity of drugs inducing OPG expression to protect bone in animal models of arthritis, in PTH induced bone resorption in rats and in metastasis of breast cancer cells to bone has been demonstrated (Onyia et al., 2004).
From the description of the biology of RANK, RANKL and OPG, it is clear that influencing the activity or differentiation of osteoclasts through modulation of these factors has potential not only in RA, but also for the treatment of osteoporosis. In addition, as bone metastasis associated with cancer also requires bone remodeling, inhibitors of osteoclast activity or differentiation could also be of use for this indication. For a review on bone metastasis, see Roodman, 2004.